BACKGROUND

The literature investigating pancreatic invasive intraductal papillary mucinous neoplasm (IPMN) has largely come from small institutional studies, preventing adequately powered comparisons of adjuvant therapy versus surgery alone (SA) within specific patient subgroups.

METHODS

Patients with resected, stage I through IV, invasive IPMN and conventional pancreatic ductal adenocarcinoma (PDAC) were identified in the National Cancer Data Base (1998-2010). Cox modeling of patients with invasive IPMN was used to compare overall survival (OS) between patients who received adjuvant therapy and those who underwent SA. A second model was used to compare OS between patients with invasive IPMN and those with PDAC.

RESULTS

For the 1220 patients with invasive IPMN, the median OS was 28.9 months; the 1-year and 5-year actuarial survival rates were 76% and 17%, respectively; and 47% received adjuvant therapy. Cox modeling associated SA with worse OS (hazard ratio, 1.36; 95% confidence interval, 1.17-1.58; P = .00005) as well as American Joint Committee on Cancer (AJCC) TNM stage III/IV disease, positive lymph node status, positive margins, and poor tumor differentiation (all P ≤ .05). In addition, Cox modeling stratified by the following characteristics demonstrated improved OS with adjuvant therapy: AJCC TNM stage II or III/IV, positive lymph node status, positive margins, and poorly differentiated tumors. There was no survival advantage from adjuvant therapy for patients who had AJCC TNM stage I or lymph node-negative disease. Patients who had invasive IPMN had improved risk-adjusted OS compared with those who had PDAC (hazard ratio, 0.73; 95% confidence interval, 0.68-0.78; P < .00001).

CONCLUSIONS

Invasive IPMN appears to be more indolent than conventional PDAC. Adjuvant therapy is associated with improved OS compared with SA in patients with invasive IPMN, especially for those with higher stage disease, positive lymph nodes, positive margins, or poorly differentiated tumors. Conversely, this benefit does not extend to patients with stage I or lymph node-negative disease.

BACKGROUND

Azzalure® (Galderma SA), a newly approved European botulinum neurotoxin type A (BoNT-A), is derived from Dysport™ (Ipsen Ltd.), which has a 20-year history of product consistency and has been widely used for various aesthetic and therapeutic applications. Azzalure® and Dysport™ are collectively referred to as BoNT-A (Speywood Unit) after the unit of their activity, and are distinct from other commercial BoNT-A preparations. Consensus has been developed for the treatment of upper facial wrinkles with BoNT-A (Speywood Unit).

OBJECTIVE

To provide consensus recommendations on the treatment with BoNT-A (Speywood Unit) for wrinkles on the middle and lower face, neck and chest region.

METHODS

The members of the International Board on Botulinum toxin Azzalure (IBBA) convened to develop consensus based on their extensive experience.

RESULTS

The recommended final concentration of BoNT-A (Speywood Unit) is 200 Speywood Units/ml after reconstitution. The consensus recommendations were provided for nine indications, including lower eyelid wrinkles, bunny lines, drooping nasal tip, perioral wrinkles, masseter hypertrophy, drooping mouth corners, dimpled chin, platysmal bands and décolleté wrinkles. For each indication, anatomy of the region to be treated was discussed, as were potential side-effects. The consensus recommendations included the number and location of the injection points, dose range of each point and the total injection, as well as specific injection technique.

CONCLUSIONS

These recommendations provide a guideline for physicians who wish to perform safe and efficacious treatment with BoNT-A (Speywood Unit) on the less commonly treated middle and lower face, neck and chest region.

The human lysosomal enzymes alpha-galactosidase (alpha-GAL, EC 3.2.1.22) and alpha-N-acetylgalactosaminidase (alpha-NAGAL, EC 3.2.1.49) share 46% amino acid sequence identity and have similar folds. The active sites of the two enzymes share 11 of 13 amino acids, differing only where they interact with the 2-position of the substrates. Using a rational protein engineering approach, we interconverted the enzymatic specificity of alpha- GAL and alpha-NAGAL. The engineered alpha-GAL (which we call alpha-GAL(SA)) retains the antigenicity of alpha-GAL but has acquired the enzymatic specificity of alpha-NAGAL. Conversely, the engineered alpha-NAGAL (which we call alpha-NAGAL(EL)) retains the antigenicity of alpha-NAGAL but has acquired the enzymatic specificity of the alpha-GAL enzyme. Comparison of the crystal structures of the designed enzyme alpha-GAL(SA) to the wild-type enzymes shows that active sites of alpha-GAL(SA) and alpha-NAGAL superimpose well, indicating success of the rational design. The designed enzymes might be useful as non-immunogenic alternatives in enzyme replacement therapy for treatment of lysosomal storage disorders such as Fabry disease.

BACKGROUND

Coenzyme Q10 (CoQ10) protects myocardium from ischemia-reperfusion (IR) injury as evidenced by improved recovery of mechanical function, ATP, and phosphocreatine during reperfusion. This protection may result from CoQ10's bioenergetic effects on the mitochondria, from its antioxidant properties, or both. The purpose of this study was to elucidate the effects of CoQ10 supplementation on mitochondrial function during myocardial ischemia-reperfusion using an isolated mitochondrial preparation.

METHODS

Isolated hearts (n = 6/group) from rats pretreated with liposomal CoQ10 (10 mg/kg iv, CoQ10), vehicle (liposomal only, Vehicle), or saline (Saline) 30 min before the experiments were subjected to 15 min of equilibration (EQ), 25 min of ischemia (I), and 40 min of reperfusion (RP). Left ventricular-developed pressure (DP) was measured. Mitochondria were isolated at end-equilibration (end-EQ), at end-ischemia (end-I), and at end-reperfusion (end-RP). Mitochondrial respiratory function (State 2, 3, and 4, respiratory control index (RCI, ratio of State 3 to 4), and ADP:O ratio) was measured by polarography using NADH (alpha-ketoglutarate, alpha-KG)- or FADH (succinate, SA)-dependent substrates.

RESULTS

CoQ10 improved recovery of DP at end-RP (67 +/- 11% in CoQ10 vs 47 +/- 5% in Vehicle and 50 +/- 11% in Saline, P < 0.05 vs Vehicle and Saline). CoQ10 did not change preischemic mitochondrial function. IR decreased State 3 and RCI in all groups using either substrate. CoQ10 had no effect in the mitochondrial oxidation of alpha-KG at end-I. CoQ10 improved State 3 at end-I when SA was used (167 +/- 21 in CoQ10 vs 120 +/- 10 in Saline and 111 +/- 10 ng-atoms O/min/mg protein in Vehicle, P < 0.05). Using alpha-KG as a substrate, CoQ10 improved RCI at end-RP (4.2 +/- 0.2 in CoQ10 vs 3.2 +/- 0.2 in Saline and 3.0 +/- 0.3 in Vehicle, P < 0.05). Using SA, CoQ10 improved State 3 (181 +/- 10 in CoQ10 vs 142 +/- 9 in Saline and 140 +/- 12 ng-atoms O/min/mg protein in Vehicle, P < 0.05) and RCI (2.21 +/- 0.06 in CoQ10 vs 1.85 +/- 0.11 in Saline and 1.72 +/- 0.08 in Vehicle, P < 0.05) at end-RP.

CONCLUSIONS

The cardioprotective effects of CoQ10 can be attributed to the preservation of mitochondrial function during reperfusion as evidenced by improved FADH-dependent oxidation.

Defective macroautophagy/autophagy and mitochondrial dysfunction are known to stimulate senescence. The mitochondrial regulator PPARGC1A (peroxisome proliferator activated receptor gamma, coactivator 1 alpha) regulates mitochondrial biogenesis, reducing senescence of vascular smooth muscle cells (VSMCs); however, it is unknown whether autophagy mediates PPARGC1A-protective effects on senescence. Using ppargc1a^-/- VSMCs, we identified the autophagy receptor SQSTM1/p62 (sequestosome 1) as a major regulator of autophagy and senescence of VSMCs. Abnormal autophagosomes were observed in VSMCs in aortas of ppargc1a^-/- mice. ppargc1a^-/- VSMCs in culture presented reductions in LC3-II levels; in autophagosome number; and in the expression of SQSTM1 (protein and mRNA), LAMP2 (lysosomal-associated membrane protein 2), CTSD (cathepsin D), and TFRC (transferrin receptor). Reduced SQSTM1 protein expression was also observed in aortas of ppargc1a^-/- mice and was upregulated by PPARGC1A overexpression, suggesting that SQSTM1 is a direct target of PPARGC1A. Inhibition of autophagy by 3-MA (3 methyladenine), spautin-1 or Atg5 (autophagy related 5) siRNA stimulated senescence. Rapamycin rescued the effect of Atg5 siRNA in Ppargc1a^+/+, but not in ppargc1a^-/- VSMCs, suggesting that other targets of MTOR (mechanistic target of rapamycin kinase), in addition to autophagy, also contribute to senescence. Sqstm1 siRNA increased senescence basally and in response to AGT II (angiotensin II) and zinc overload, two known inducers of senescence. Furthermore, Sqstm1 gene deficiency mimicked the phenotype of Ppargc1a depletion by presenting reduced autophagy and increased senescence in vitro and in vivo. Thus, PPARGC1A upregulates autophagy reducing senescence by a SQSTM1-dependent mechanism. We propose SQSTM1 as a novel target in therapeutic interventions reducing senescence. Abbreviations: 3-MA: 3 methyladenine; ACTA2/SM-actin: actin, alpha 2, smooth muscle, aorta; ACTB/β-actin: actin beta; AGT II: angiotensin II; ATG5: autophagy related 5; BECN1: beclin 1; CAT: catalase; CDKN1A: cyclin-dependent kinase inhibitor 1A (P21); Chl: chloroquine; CTSD: cathepsin D; CYCS: cytochrome C, somatic; DHE: dihydroethidium; DPBS: Dulbecco's phosphate-buffered saline; EL: elastic lamina; EM: extracellular matrix; FDG: fluorescein-di-β-D-galactopyranoside; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; γH2AFX: phosphorylated H2A histone family, member X, H₂DCFDA: 2',7'-dichlorodihydrofluorescein diacetate; LAMP2: lysosomal-associated membrane protein 2; MASMs: mouse vascular smooth muscle cells; MEF: mouse embryonic fibroblast; NBR1: NBR1, autophagy cargo receptor; NFKB/NF-κB: nuclear factor of kappa light polypeptide gene enhancer in B cells; MTOR: mechanistic target of rapamycin kinase; NFE2L2: nuclear factor, erythroid derived 2, like 2; NOX1: NADPH oxidase 1; OPTN: optineurin; PFA: paraformaldehyde; PFU: plaque-forming units; PPARGC1A/PGC-1α: peroxisome proliferator activated receptor, gamma, coactivator 1 alpha; Ptdln3K: phosphatidylinositol 3-kinase; RASMs: rat vascular smooth muscle cells; ROS: reactive oxygen species; SA-GLB1/β-gal: senescence-associated galactosidase, beta 1; SASP: senescence-associated secretory phenotype; SIRT1: sirtuin 1; Spautin 1: specific and potent autophagy inhibitor 1; SQSTM1/p62: sequestosome 1; SOD: superoxide dismutase; TEM: transmission electron microscopy; TFEB: transcription factor EB; TFRC: transferrin receptor; TRP53/p53: transformation related protein 53; TUBG1: tubulin gamma 1; VSMCs: vascular smooth muscle cells; WT: wild type.

The rapidly inactivating (A-type) potassium channels regulate membrane excitability that defines the fundamental mechanism of neuronal functions such as pain signaling. Cytosolic Kv channel-interacting proteins KChIPs that belong to neuronal calcium sensor (NCS) family of calcium binding EF-hand proteins co-assemble with Kv4 (Shal) alpha subunits to form a native complex that encodes major components of neuronal somatodendritic A-type K+ current, I(SA), in neurons and transient outward current, I(TO), in cardiac myocytes. The specific binding of auxiliary KChIPs to the Kv4 N-terminus results in modulation of gating properties, surface expression and subunit assembly of Kv4 channels. Here, I attempt to emphasize the interaction between KChIPs and Kv4 based on recent progress made in understanding the structure complex in which a single KChIP1 molecule laterally clamps two neighboring Kv4.3 N-termini in a 4:4 manner. Greater insights into molecular mechanism between KChIPs and Kv4 interaction may provide therapeutic potentials of designing compounds aimed at disrupting the protein-protein interaction for treatment of membrane excitability-related disorders.

Phagocytes play a major role in host defense against staphylococci as well as in the pathophysiology of Gram-positive septic shock. In Gram negative sepsis, the main mediator, LPS exerts its effects as easily suspendable mediator. In Gram positive sepsis the main mediator is still not found, therefore we studied the interaction of soluble staphylococcal products with phagocytes. Staphylococcus aureus supernates (SaS) were harvested from several laboratory and clinical strains that were grown to late-log phase. These supernates upregulated CD11b/CD18 expression on human neutrophils even in a 100-fold dilution. SaS also induced the release of TNF-alpha and IL-1 beta by human monocytes. Control experiments excluded peptidoglycan, lipoteichoic acid, alpha and delta toxin, leucocidin, TSST-1 and all enterotoxins as sole mediators. Endotoxin contamination was also excluded. SaS was heat-stable; incubation for 45 minutes at 100 degrees C did not affect its activity. Compared to purified peptidoglycan and intact bacteria per bacterium, SaS had a higher potency in stimulating phagocytes. We hypothesize that there are more--yet unknown--soluble staphylococcal products which are very important in phagocyte stimulation.

To elucidate control mechanisms of O-glycan biosynthesis in leukemia and to develop biosynthetic inhibitors we have characterized core 2 UDP-GlcNAc:Gal beta 1-3GalNAc-R(GlcNAc to GalNAc) beta 6-N-acetylglucosaminyltransferase (EC 2.4.1.102; core 2 beta 6-GlcNAc-T) and CMP-sialic acid: Gal beta 1-3GalNAc-R alpha 3-sialyltransferase (EC 2.4.99.4; alpha 3-SA-T), two enzymes that are significantly increased in patients with chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML). We observed distinct tissue-specific kinetic differences for the core 2 beta 6-GlcNAc-T activity; core 2 beta 6-GlcNAc-T from mucin secreting tissue (named core 2 beta 6-GlcNAc-T M) is accompanied by activities that synthesize core 4 [GlcNAc beta 1-6(GlcNAc beta 1-3)GalNAc-R] and blood group I [GlcNAc beta 1-6(GlcNAc beta 1-3)Gal beta-R] branches; core 2 beta 6-GlcNAc-T in leukemic cells (named core 2 beta-GlcNAc-T L) is not accompanied by these two activities and has a more restricted specificity. Core 2 beta 6-GlcNAc-T M and L both have an absolute requirement for the 4- and 6-hydroxyls of N-acetylgalactosamine and the 6-hydroxyl of galactose of the Gal beta 1-3GalNAc alpha-benzyl substrate but the recognition of other substituents of the sugar rings varies, depending on the tissue. alpha 3-sialyltransferase from human placenta and from AML cells also showed distinct specificity differences, although the enzymes from both tissues have an absolute requirement for the 3-hydroxyl of the galactose residue of Gal beta 1-3GalNAc alpha-Bn. Gal beta 1-3(6-deoxy)GalNAc alpha-Bn and 3-deoxy-Gal beta 1-3GalNAc alpha-Bn competitively inhibited core 2 beta 6-GlcNAc-T and alpha 3-sialyltransferase activities, respectively.

The continuously high prevalence of methicillin-resistant staphylococci (MRS) throughout the world is a constant threat to public health, owing to the multiresistant characteristics of these bacteria. Methicillin resistance is phenotypically associated with the presence of the penicillin-binding protein 2a (PBP2a) not present in susceptible staphylococci. This protein has a low binding affinity for beta-lactam antibiotics. It is a transpeptidase which may take over cell wall synthesis during antibiotic treatment when normally occurring PBPs are inactivated by ligating beta-lactams. PBP2a is encoded by the mecA gene, which is located in mec, a foreign DNA region. Expression of PBP2a is regulated by proteins encoded by the plasmid-borne blaR1-bla1 inducer-repressor system and the corresponding genomic mecRl-mecl system. The blaRl-blal products are important both for the regulation of beta-lactamase and for mecA expression. Methicillin resistance is influenced by a number of additional factors, e.g. the products of the chromosomal fem genes which are important in the synthesis of normal peptidoglycan precursor molecules. Inactivation of fem-genes results in structurally deficient precursors which are not accepted as cell wall building blocks by the ligating PBP2a transpeptidase during antibiotic treatment. This may result in reduced resistance to beta-lactam antibiotics. Inactivation of genes affecting autolysis has shown that autolytic enzymes are also of importance in the expression of methicillin resistance. Methicillin resistance has evolved among earth microorganisms for protection against exogenous or endogenous antibiotics. Presumably the mec region was originally transferred from coagulase negative staphylococci (CNS) to Staphylococcus aureus (SA). A single or a few events of this kind with little subsequent interspecies transfer had been anticipated. However, recent data suggest a continuous horizontal acquisition by S. aureus of mec, being unidirectional from CNS to SA. Methicillin resistance may also be associated with mechanisms independent of mecA, resulting in borderline methicillin resistance. These mechanisms include beta-lactamase hyperproduction, production of methicillinases, acquisition of structurally modified normal PBPs, or the appearance of small colony variants of SA. Most MRS are multiresistant, and the mec region may harbour several resistance determinants, resulting in a clustering of resistance genes within this region.

Dried blood spot (DBS) samples on filter paper are surging in popularity as a sampling and storage vehicle for a wide range of clinical and pharmaceutical applications. For example, a DBS sample is collected from every baby born in the province of Ontario, Canada, for quantification of approximately one hundred analytes that are used to screen for 28 conditions, including succinylacetone (SA), a marker for hepatorenal tyrosinemia. Unfortunately, the conventional methods used to evaluate DBS samples for newborn screening and other applications are tedious and slow, with limited options for automated analysis. In response to this challenge, we have developed a method to couple digital microfluidics (DMF) to nanoelectrospray ionization mass spectrometry (nESI-MS) for SA quantification in DBS samples. The new system is formed by sandwiching a pulled glass capillary emitter between the two DMF substrates such that the capillary emitter is immobilized without external seals or gaskets. Moreover, we introduce a new feedback control system that enables high-fidelity droplet manipulation across DBS samples without manual intervention. The system was validated by application to on-chip extraction, derivatization, and analysis of SA and other analytes from DBS samples, with comparable performance to gold-standard methods. We propose that the new methods described here can potentially contribute to a new generation of analytical techniques for quantifying analytes in DBS samples for a wide range of applications.

The POU DNA-binding motif defines a conserved family of eukaryotic transcription factors involved in regulation of gene expression. This bipartite motif consists of an N-terminal POU-specific domain (POUs), a flexible linker, and a C-terminal POU-specific homeodomain (POUHD). Here we describe the solution structure of a POU-specific homeodomain. An NMR model is obtained from Oct-2, a human B-cell specific transcription factor which participates in the regulation of immunoglobulin genes. A fragment of Oct-2 containing POUHD and an adjoining linker was expressed in Escherichia coli and characterized by three-dimensional nuclear magnetic resonance (3D-NMR) spectroscopy. Complete 1H and 15N resonance assignment of the POUHD moiety is presented. The POUHD solution structure, as calculated by distance geometry and simulated annealing (DG/SA), is similar to that of canonical homeodomains. A salient difference between solution and crystal structures is observed in the C-terminal segment of alpha-helix 3 (the HTH recognition helix), which is not well ordered in solution. Because this segment presumably folds upon specific DNA binding, its flexibility in solution may reduce the intrinsic DNA affinity of POUHD in the absence of POUs.

The role of the interplay of interleukin-2 (IL-2) and interleukin-10 (IL-10) in responses of human peripheral blood B cells stimulated by ligation of antigen receptors was examined in detail. Highly purified peripheral blood B cells from normal human individuals were cultured with Staphylococcus aureus Cowan I (SA) in the presence or absence of IL-10 and IL-2. Although IL-10 alone could modestly induce Ig production by SA-activated B cells, IL-10 markedly enhanced the Ig production in synergism with IL-2. This synergistic effect between IL-2 and IL-10 was completely abrogated by addition of either anti-CD25 mAb or anti-IL-10 mAb, indicating signals through high-affinity IL-2 receptors are involved in the synergism. In fact, IL-10 upregulated the expression of alpha-chain of IL-2 receptors (CD25) on B cells stimulated by SA alone or SA + IL-2 for 72 hr. However, such upregulation of CD25 expression did not result in significant enhancement of subsequent responses of the activated B cells to IL-2. Of note, during the initial activation phase of B cells, IL-2 was much more effective than IL-10 in enhancing the subsequent responses to either IL-2 or IL-10. By contrast, IL-10 was much more effective than IL-2 in supporting the maturation of B cells following the initial activation. Finally, the synergy between IL-2 and IL-10 was not observed in the initial activation phase, but in the subsequent maturation phase of activated B cells. These results indicate that IL-10 and IL-2 synergistically enhance Ig production of SA-activated B cells in a mechanism which is different from the upregulation of IL-2 receptors. Moreover, the data emphasize the importance of the interplay of IL-2 and IL-10 in determining the outcome of humoral immune responses.

Statins affect the production of long chain polyunsaturated fatty acids (PUFA), both in vitro and in vivo. Various studies have shown the effects of statins on the pattern of n-6 fatty acids (FA), but limited attention has been paid to the n-3 FA. We investigated, in THP-1 and in HepG2 cells, the effects of simvastatin on the conversion of the 18C FA precursors in the n-3 and n-6 series, [1-(14)C] alpha-linolenic acid (alpha-LNA) and [1-(14)C] linoleic acid (LA) respectively, and on the metabolism of [1-(14)C] stearic acid (SA). THP-1 cells, as in the case of LA, actively converted alpha-LNA to its products, and after simvastatin treatment, the total conversion was significantly increased (from 57.2+/-7.2 to 74.3+/-8.5%, p<0.05). HepG2 cells also converted LA and alpha-LNA, but simvastatin increased significantly only the conversion of LA (9.5+/-1.9% versus 23.8+/-5.1%, p<0.02). SA conversion was similar in untreated cells (about 50%), while statin increased the production of oleic acid in HepG2, but in THP-1 cells there was a decrease. In conclusion, LA, alpha-LNA and SA are differentially metabolized in THP-1 and in HepG2 cells and their increased conversion by simvastatin is lower in HepG2 than in THP-1. These differences may reflect the distinct features of the two cell lines: monocytes, precursors of phagocytic cells, versus hepatocytes with mainly metabolic functions. Substantial differences concern also cellular FA pools: structural in THP-1 cells, and also depot, resulting in sequestering of the substrates, in HepG2. The greater n-3 FA metabolism in THP-1 cells may have favourable functional effects.

Increasing numbers of H5N1 influenza viruses (IVs) are responsible for human deaths, especially in North Africa and Southeast Asian. The binding of hemagglutinin (HA) on the viral surface to host sialic acid (SA) receptors is a requisite step in the infection process. Phylogenetic analysis reveals that H5N1 viruses can be divided into 10 clades based on their HA sequences, with most human IVs centered from clade 1 and clade 2.1 to clade 2.3. Protein sequence alignment in various clades indicates the high conservation in the receptor-binding domains (RBDs) is essential for binding with the SA receptor. Two glycosylation sites, 158N and 169N, also participate in receptor recognition. In the present work, we attempted to construct a serial H5N1 HA models including diverse glycosylated HAs to simulate the binding process with various SA receptors in silico. As the SA-α-2,3-Gal and SA-α-2,6-Gal receptor adopted two distinctive topologies, straight and fishhook-like, respectively, the presence of N-glycans at 158N would decrease the affinity of HA for all of the receptors, particularly SA-α-2,6-Gal analogs. The steric clashes of the huge glycans shown at another glycosylation site, 169N, located on an adjacent HA monomer, would be more effective in preventing the binding of SA-α-2,3-Gal analogs.

The replicative life span of cells in culture is thought to be determined by the gradually rising pool of senescent cells rather than by the simultaneous loss of proliferative capacity by all cells in the population. We found that early-passage cultures of human peritoneal mesothelial cells (HPMCs) contained a significant fraction of senescent-like cells. Furthermore, early-passage populations with a high percentage of senescent cells had a reduced subsequent life span in culture compared with populations consisting of the same number of apparently young cells but containing no senescent cells. The exposure of early-passage HPMCs to the conditioned medium from cultures containing senescent cells resulted in the retardation of growth and the induction of senescence-associated beta-galactosidase (SA-beta-Gal). This effect could be partly reduced by neutralizing TGF-beta1 activity. The timely treatment with N-tert-butyl-alpha-phenylnitrone (PBN) reduced oxidative stress, the number of early senescent cells, TGF-beta1 secretion, and ultimately extended the population life span. The effect was evident only when PBN was introduced at a very early, but not at a late, phase of tissue culture history. These results indicate that a sudden onset of senescence in early-passage HPMCs is related to oxidative stress and may influence the replicative life span of the population as a whole.

BACKGROUND

Bio-manufacturing of high-value chemicals in parallel to renewable biofuels has the potential to dramatically improve the overall economic landscape of integrated lignocellulosic biorefineries. However, this will require the generation of carbohydrate streams from lignocellulose in a form suitable for efficient microbial conversion and downstream processing appropriate to the desired end use, making overall process development, along with selection of appropriate target molecules, crucial to the integrated biorefinery. Succinic acid (SA), a high-value target molecule, can be biologically produced from sugars and has the potential to serve as a platform chemical for various chemical and polymer applications. However, the feasibility of microbial SA production at industrially relevant productivities and yields from lignocellulosic biorefinery streams has not yet been reported.

RESULTS

Actinobacillus succinogenes 130Z was immobilised in a custom continuous fermentation setup to produce SA on the xylose-enriched fraction of a non-detoxified, xylose-rich corn stover hydrolysate stream produced from deacetylation and dilute acid pretreatment. Effective biofilm attachment, which serves as a natural cell retention strategy to increase cell densities, productivities and resistance to toxicity, was accomplished by means of a novel agitator fitting. A maximum SA titre, yield and productivity of 39.6 g L(-1), 0.78 g g(-1) and 1.77 g L(-1) h(-1) were achieved, respectively. Steady states were obtained at dilution rates of 0.02, 0.03, 0.04, and 0.05 h(-1) and the stirred biofilm reactor was stable over prolonged periods of operation with a combined fermentation time of 1550 h. Furthermore, it was found that a gradual increase in the dilution rate was required to facilitate adaptation of the culture to the hydrolysate, suggesting a strong evolutionary response to the toxic compounds in the hydrolysate. Moreover, the two primary suspected fermentation inhibitors, furfural and HMF, were metabolised during fermentation with the concentration of each remaining at zero across all steady states.

CONCLUSIONS

The results demonstrate that immobilised A. succinogenes has the potential for effective conversion of an industrially relevant, biomass-derived feed stream to succinic acid. Furthermore, due to the attractive yields, productivities and titres achieved in this study, the process has the potential to serve as a means for value-added chemical manufacturing in the integrated biorefinery.

The molecular basis of the potassium ion (K+) channels that generate repolarization in heart tissue remains uncertain, in part because of the molecular diversity of the voltage-gated K+ channel family. In our investigation, we used fluorescent labeled oligonucleotide probes to perform in situ hybridization studies on enzymatically isolated myocytes to determine the identity, regional distribution, and cellular distribution of voltage-gated K+ channel, alpha-subunit mRNA expressed in ferret heart. The regions studied were from the sinoatrial node (SA), right and left atrium, right and left ventricle, and interatrial and interventricular septa. Kv1.5 and Kv1.4 were the most widely distributed K+ channel transcripts in the ferret heart (present in approximately 70%-86% and approximately 46%-95% of tested myocytes, respectively), followed by Kv1.2, Kv2.1, and Kv4.2. In addition, many myocytes contain transcripts for Kv1.3, Kv2.2, Kv4.1, Kv5.1, and members of the Kv3 family. Kv1.1, Kv1.6, and Kv6.1 were rarely expressed in working myocytes, but were more commonly expressed in SA nodal cells. Two other transcripts whose genes have been implicated in the long QT syndrome, erg and KvLQT1, were common in all regions (approximately 41%-58% and 52%-72%, respectively). These results show that both the diversity and heterogeneity of K+ channel mRNA in heart tissue is greater than previously suspected.

The first liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for determination of acetylsalicylic acid (aspirin, ASA) and one of its major metabolites, salicylic acid (SA), in human plasma using simvastatin as an internal standard has been developed and validated. For ASA analysis, a plasma sample containing potassium fluoride was extracted using a mixture of ethyl acetate and diethyl ether in the presence of 0.5% formic acid. SA, a major metabolite of ASA, was extracted from plasma using protein precipitation with acetonitrile. The compounds were separated on a reversed-phase column with an isocratic mobile phase consisting of acetonitrile and water containing 0.1% formic acid (8:2, v/v). The ion transitions recorded in multiple reaction monitoring mode were m/z 179 ->> 137, 137 ->> 93 and 435 ->> 319 for ASA, SA and IS, respectively. The coefficient of variation of the assay precision was less than 9.3%, and the accuracy exceeded 86.5%. The lower limits of quantification for ASA and SA were 5 and 50 ng/mL, respectively. The developed assay method was successfully applied for the evaluation of pharmacokinetics of ASA and SA after single oral administration of Astrix (entero-coated pellet, 100 mg of aspirin) to 10 Korean healthy male volunteers.

With the aim of improving the chemotherapeutic index of 9-beta-D-arabinofuranosyl-adenine 5' monophosphate (ara-AMP) in the treatment of chronic hepatitis B, this drug was conjugated with lactosaminated serum albumin (L-SA), a neoglycoprotein which only enters into hepatocytes. We used a L-SA-ara-AMP conjugate which, in contrast to those previously employed, has the advantage of remaining soluble after lyophilization. We found in mice that: (I) this new conjugate was quite stable in the bloodstream where only a small part of ara-AMP was released; (II) after administration of the conjugate labelled in the drug moiety both acid insoluble and soluble radioactivities were several times higher in liver than in other organs; (III) in mice with Ectromelia virus hepatitis, the conjugate inhibited virus DNA synthesis in liver without affecting cellular DNA synthesis in intestine and bone marrow; (IV) the conjugate did not display any recognizable sign of acute toxicity even at doses several fold higher than those pharmacologically active; and (V) when prepared with homologous albumin it was not immunogenic.

Effects of phorbol esters on spontaneously beating rabbit sino-atrial (SA) node cells were investigated by means of voltage clamp technique. In a small SA node specimen, 12-O-tetradecanoylphorbol-13-acetate (TPA) 10(-7) mol/l lengthened the cycle length (CL) and at over 3 X 10(-7) mol/l prolonged the action potential duration (APD). Action potential amplitude (APA), maximum diastolic potential (MDP) and maximum rate of rise (Vmax) were unaffected. Amiloride 10(-3) mol/l, an inhibitor of Na+-H+ exchange, did not reverse the phorbol ester-induced effects. In voltage-clamp experiments, TPA 1-10 X 10(-7) mol/l slightly increased the slow inward current (Isi) and the time-dependent inward current (Ih) which activates during hyperpolarization. The outward current and the tail current were reduced, although the activation curve was not shifted along the voltage axis. In the presence of 10(-7) mol/l isoprenaline, TPA produced dysrhythmia and a transient inward current in voltage-clamp experiments. In the presence of 5 X 10(-5) mol/l phenylephrine or 2 X 10(-6) mol/l acetylcholine, TPA also elicited dysrhythmia. 4-beta-phorbol-12,13-dibutyrate (PBD) induced similar electrophysiological effects as TPA, but 4-alpha-phorbol-12,13-didecanoate (PDD) never did so even in the presence of isoprenaline. These results suggest that TPA and PDB might mobilize intracellular Ca2+ via protein kinase C activation in the presence of isoprenaline, phenylephrine or acetylcholine, resulting in dysrhythmia due to delayed afterdepolarization.

OBJECTIVE

It is believed that the magnitude of the systemic inflammatory response induced by percutaneous coronary intervention (PCI) impacts on the long-term outcomes in patients with stable angina (SA) and unstable angina (UA). We aimed to determine whether an inflammatory response appears in in-stent restenosis (ISR) patients undergoing balloon angioplasty and to assess its pattern and magnitude in relation to SA and UA subjects.

METHODS

80 patients (59 with SA, 10 with UA, 11 with ISR) were enrolled into the prospective study.

METHODS

SA and UA patients undergoing single vessel coronary balloon angioplasty followed by stenting versus ISR subjects in whom only balloon angioplasty was performed.

METHODS

C-reactive protein (CRP), serum amyloid A (SAA), tumor necrosis factor alpha (TNF-alpha) and interleukin 10 (IL-10) were measured in blood samples collected before and 6, 24 h and 1 month after the procedure.

RESULTS

A comparable pattern of inflammatory response in terms of CRP and SAA concentrations in subjects undergoing PCI due to ISR and SA was discovered while in unstable patients its magnitude was substantially higher. CRP and SAA levels increased significantly in each group with the peak value at 24 h and the baseline levels remarkably correlated with the highest markers' concentrations. In contrast, preprocedural TNF-alpha concentrations were higher in ISR group when compared with SA and UA patients. Additionally, in ISR group a twofold increase in their values of borderline significance at 6 h was noted. SA and UA subjects were found to have significantly lower TNF-alpha levels at 6 and 24 h after the intervention though the marker concentrations markedly increased with peak values at 1 month. The levels of IL-10 did not differ at any time point between the groups.

CONCLUSIONS

We suggest that PCI triggers a systemic inflammatory response in patients with ISR and considerable differences in its pattern when compared with SA and UA patients were demonstrated. Moreover, a high preprocedural TNF-alpha level and its increase provoked by PCI in the ISR group warrant the need for further investigation of its possible involvement in the restenosis process.

Investigating the empirical support of various assessment instruments, the evidence based assessment approach expands the scientific basis of psychotherapy. Starting from Hunsley and Mash's evaluative framework, we critically reviewed the rating scales designed to measure social anxiety or phobia in youth. Thirteen of the most researched social anxiety scales for children and adolescents were identified. An overview about the scientific support accumulated by these scales is offered. Our main results are consistent with recent reviews that consider the Social Phobia and Anxiety Scale for Children (SPAI-C) and the Social Anxiety Scale for Adolescents (SAS-A) among the most pertinent and empirically supported measures of social anxiety for youngsters. However, after considering the existing evidence, we highly recommend another couple of scales that proved to be empirically supported (i.e., the Social Phobia Inventory-SPIN, and the Liebowitz Social Anxiety Scale for Children and Adolescents-LSAS-CA).

SAs, a structurally related group of antibiotics containing a similar 4-aminobenzene sulfonamide backbone, are used in agriculture, aquaculture, animal husbandry, and also as human medicines. Competing with p-aminobenzoic acid in the enzymatic synthesis of dihydrofolic acid, SAs inhibit the growth and reproduction of bacteria. Once released to the environment, SAs distribute themselves among different environmental compartments, along with their degradation products, and are transported to surface water and groundwater. The physicochemical properties, the dosage applied and the nature of the environmental components with which they interact, govern the whole process. SAs, as a class, are less sorptive, impersistent, and leachable. They cannot be characterized as readily biodegradable. Their adsorption to soil increases with the aromaticity and electronegativity of functional groups attached to the sulfonyl phenyl amine core. Preferential flow in clay soils has been identified as a mechanism responsible for surface water contamination by SAs.

BACKGROUND

Some previously published research on treatment utilization patterns in patients with attention deficit/hyperactivity disorder (ADHD) has been focused on data from commercial health plans, whereas research in the Medicaid population is lacking. Thus, little is known about these utilization patterns in Medicaid populations, which typically have demographic and clinical characteristics that differ from those of employer-based groups.

OBJECTIVE

The objectives of the present retrospective data analysis were to evaluate the associations of medication groups (categorized by stimulant type [methylphenidate or amphetamine] and duration of action [short-acting (SA) or long-acting (LA)]) with measures of stimulant utilization patterns (adherence, persistence, and switching) in children, adolescents, and adults with ADHD enrolled in the fee-for-service delivery model within the Texas Medicaid Program.

METHODS

Texas Medicaid fee-for-service claims data were analyzed retrospectively. Data from enrollees with ADHD (6-63 years) were included if patients were newly initiated on medication from January 2006 to September 2007, had ≥1 medical claim with a code for ADHD, and had continuous Medicaid eligibility 6 months before and after treatment initiation. Adherence, persistence, and switching were compared by initial stimulant medication group (SA methylphenidate [SA-M], LA-M, SA amphetamine [SA-A], and LA-A). Rates were compared overall and by age group (children, adolescents, and adults). Multivariate models were used to control for demographic, clinical, and utilization covariates.

RESULTS

Of 15,055 eligible patients, mostly children, 71% were initiated on methylphenidate; 90% received LA formulations (LA-M, 65%; LA-A, 25%). Most children (66%) and adolescents (65%) were initiated on LA-M, followed by LA-A (23% and 29%, respectively). Among adults, 38% each were initiated on LA-M and LA-A. Overall adherence (measured using the days in possession ratio [DPR]) and persistence were significantly greater with the LA versus the SA formulations (mean DPR, 0.497-0.504 vs 0.407-0.418, respectively; mean persistence, 81 vs 66-67 days; both, P < 0.001), and the rates of switching were lower with the LA versus the SA formulations (12.3%-14% vs 27%-28%; P < 0.001). On multivariate analyses, the likelihoods of adherence and persistence were significantly greater with the LA formulations, and the likelihood of being switched was significantly greater with the SA formulations (P < 0.001). These analyses also showed that medication utilization was significantly related to demographic and clinical characteristics.

CONCLUSIONS

Based on the findings from this retrospective analysis, ADHD treatment utilization patterns varied by formulation in this Texas Medicaid population. Persistence at 180 days was poor regardless of the stimulant used. Consideration of stimulant formulations and demographic characteristics in patients in whom long-term ADHD management is being initiated may assist in optimum utilization, perhaps leading to better symptom control and more efficient resource utilization.